The invention relates to xylanase activity (XA) proteins and nucleic acids. The invention further relates to the use of the XA proteins in the bleaching process of pulp and in the food and animal feed industry.
Glycosyl hydrolase enzymes have been classified into more than 60 families that include xylanases (Xyn), cellulases, mannanases, amylases, beta-glucanases, and other carbohydrases [Henrissat, Biochem. J. 280:309-316 (1991); Henrissat and Bairoch, Biochem. J. 293:781-788 (1993); Henrissat and Bairoch, Biochem. J. 316:695-696 (1996); Davies and Henrissat, Structure 3:853-859 (1995); Coutinho and Henrissat, in Genetics, Biochemistry and Ecology of Cellulose Degradation, eds. Ohmiya et al., Uni Publishers Co., Tokyo, pp 15-23 (1999)]. These enzymes are classified based on amino acid sequence, the three dimensional structure and the geometry of the catalytic site [Gilkes et al., Microbiol. Reviews 55:303-315 (1991)]. Xylanases are produced by many organisms of bacterial and fungal origin (Enzymes for Pulp and Paper Processing, eds. Jeffries and Viikari; ACS Symposium Series Vol.655, American Chemical Society, Washington, D.C. (1996); and Xylans and Xylanases, eds. Visser et al., Progress in Biotechnology Vol. 7; Amsterdam-London-New York-Tokyo (1992)] and are used to hydrolyze the polysaccharide xylan, which is a major component of the plant cell walls [Hemicellulose and Hemicellulases, eds. Coughlan and Hazelwood; Portland Press Ltd, London-Chapel Hill, (1993)].
The endo-beta-1,4-xylanases (EC 3.2.1.8) belong either to the family 10 xylanases, formerly known as F, or to the family 11 xylanases, also known as G. The family 10 have an (xcex1/xcex2)8 barrel fold [Dominguez et al., Nat. Struc. Biol. 2:29-35 (1995)], whereas the family 11 xylanases are mostly xcex2-sheet and the overall structure resembles that of a right hand [Torronen et al., EMBO J. 13:2493-2501 (1994)]. The Bacillus circulans xylanase belongs to the family 11.
Family 11 xylanases have been reported from varies microorganism (bacteria, yeast and fungi), including Aspergillus awamori var. kawachi xyn A [Ito, Swiss prot. Entry P48824]; Aspergillus niger Xyn A [Krengel and Dijkstra, J. Mol. Biol. 263(1):70-78 (1996); PDB entry 1 ukr]; Aspergillus kawachii Xyn C [Ito et al., Biosci. Biotechnol. Biochem. 56(8):1338-1340 (1992)]; Aspergillus tubigensis Xyn A [de Graaff et al., Mol. Microbiol. 12(3):479-490 (1994)]; Bacillus circulans Xyn A [Yang et al., Nucl. Acids. Res., 16:7187 (1988)]; Bacillus pumilus Xyn A [Fukusaki et al., FEBS Lett. 171:197-201 (1984); Bacillus subtilis Xyn A [Paice et al., Arch, Microbiol. 144:201-202 (1986)]; Bacillus sp. strain 41M-1 [Ryuichiro et al., Nucl. Acids Symp. Series 31:235-236 (1994)]; Cellulomonas fimi Xyn D; Chainia spp. Xyn; Clostridium acetobutylicum Xyn B [Zappe et al., Nucl. Acids Res. 18(8):2179 (1990); Clostridium stercorarium; Xyn A [Sakka et al., Biosci. Biotechnol. Biochem. 57(2):273-277 (1993)]; Cochliobolus carbonum [Apel et al., Mol. Plant Microbe Interact. 6(4):467-473 (1993)]; Fibrobacter succinogenes Xyn C [Paradis et al., J. Bacteriol. 175(23):7666-7672 (1993)]; Neocallimastix patriciarum Xyn A [Gilbert et al., Mol. Microbiol. 6(15):2065-2072 (1992)]; Nocardiopsis dassonvillei Xyn II; Paecilomyces varotii [J. Mol. Biol. 243(4):806-808 (1994); PDB entry 1 PVX]; Ruminococcus flavefaciens Xyn A Zhang and Flint, Mol. Microbiol. 6(8):1013-1023 (1992)]; Schizophyllum commune Xyn [Yaguchi et al., in Xylans and Xylanases, eds. Visser et al., Progress in Biotechnology Vol. 7; pp 149-154, Amsterdam-London-New York-Tokyo (1992); Streptomyces lividans Xyn B [Shareck et al., Gene 107(1):75-82 (1991)]; Streptomyces lividans Xyn C [Shareck et al., Gene 107(1):75-82 (1991)]; Streptomyces sp. No. 36a Xyn [Nagashima et al., Trends Actinomycetoligia 91-96 (1989)]; Streptomyces thermoviolaceus Xyn II; Thermomonospora fusca Xyn A; Thermomyces lanuginosus [Gruber et al., Biochemistry 37(39):13475-13485 (1998)]; Trichoderma harzianum Xyn [Campbell et al., PDB entry 1XND]; Trichoderma reesei Xyn I [Torronen and Rouvinen, Biochemistry 34:847 (1995); PDB entry 1XYN]; Trichoderma reesei Xyn II [Torronen et al., EMBO J. 13(11):2493-2501 (1994); PDB entry 1 ENX]; Trichoderma viride Xyn [Yaguchi, GenBank accession #A44594; (gi:627019)].
In recent years, xylanases have become more and more used in the pulp and paper industry in a process called kraft pulp bleaching [Enzymes for Pulp and Paper Processing, eds. Jeffries and Viikari; ACS Symposium Series Vol. 655, American Chemical Society, Washington, D.C. (1996)]. These enzymes are added to the pulp before the pulp is bleached, to enhance the bleaching process and to remove a portion of the xylan in the pulp [Paice and Jurasek, J. Wood Chem. Tech. 4(2):187-198 (1984)]. This enzymatic pre-treatment allows the subsequent bleaching chemicals, including chlorine, chlorine dioxide, hydrogen peroxide, oxygen, ozone, and sodium hydroxide, to bleach the pulp more efficiently than in the absence of xylanase treatment. The enhanced efficiency of bleaching has allowed mills to reduce the amount of chlorine-based chemicals used, thereby decreasing the amount of toxic by-products, which are environmental pollutants. In addition, less bleaching chemicals are used, lowering the chemical costs.
The Family 11 xylanases have several advantages over other xylanases in pulp bleaching applications. Most of the Family 11 xylanases are smaller than xylanases in other families. The small size relative to other xylanases is probably beneficial in penetrating the pulp fibers to release xylan from the pulp and enhance the bleaching. The Family 11 xylanases are also xe2x80x9cpurexe2x80x9d xylanases in terms of their catalytic activity. Unlike the xylanase enzymes in other families, these enzymes hydrolyze only xylan and do not hydrolyze cellulose. Cellulose hydrolysis damages the pulp and is unacceptable in a commercial mill.
In spite of the advantages of Family 11 xylanases in pulp bleaching, these enzymes have significant drawbacks. The range of temperature and pH that the enzymes exhibits activity on pulp are 45xc2x0 C. to 55xc2x0 C. and pH 5.0 to 7.5. A small proportion of mills have operated historically within these ranges. However, the step in the process where xylanase is applied is after a hot alkali treatment, so that the pulp is very basic and hot, typically having a temperature of 60xc2x0 C. to 70xc2x0 C. and a pH of 10 to 12. Both of these conditions are sub-optimal for xylanase enzymatic activity. For example, the Bacillus circulans wild type xylanase has a temperature optimum of 55xc2x0 C. and a pH optimum of 5.5. In some mills the adjustment of temperature and pH are acceptable and routine, albeit energy intensive and costly. In many mills achieving the desired treatment conditions causes severe problems. Therefore, the intrinsic properties of the enzyme, such as thermostability and activity at elevated pH are critical parameters for their use in the bio-bleaching processes.
Among naturally occurring xylanases, thermostable enzymes have been isolated from thermophilic microbes, such as Caldocellum saccharolyticum, Thermatoga maritima and Thermatoga sp. strain FjSS3-B.1, all of which grow at 80xc2x0 C. to 100xc2x0 C. [(Luthi et al. Appl. Environ. Microbiol. 56:2677-2683 (1990); Winterhalter and Liebl, Appl. Environ. Microbiol. 61:1810-1815 (1995); Simpson et al., Biochem. J. 277:413-417 (1991)]. However, all are relatively large in size with high molecular mass of 35-120 kDa (320-1100 residues) and as such, their penetration into the pulp fibers might be limited. Some of these xylanases (e.g., C. saccharolyticum xylanase A) belong to families other than Family 11, and have both xylanase and cellulase activities (Luthi, et al., supra). Such cellulase activity is undesirable for pulp bleaching. Furthermore, hyperthermostable xylanases which function normally at extremely high temperatures have low activity at the comparatively lower temperatures for pulp bleaching.
It is well known in the art that xylanase also has uses in non-pulp applications. For example, xylanases have been reported to be useful in clarifying juice and wine [Zeikus et al., ACS Symp. Ser. 460:36-51 (1991); Beily, ACS Symp. Ser. 460:408-416 (1991); Woodward, Top Enzyme Ferment. Biotechnol. 8:9-30 (1984)]; extracting coffee, plant oils and starch [Beily, supra; Woodward supra; McCleary, Int. J. Biol. Macromol. 8:349-354 (1986)]; for the production of food thickeners (Zeikus et al., supra); altering texture in bakery products, e.g., improving the quality of dough, help bread rise and processing of wheat and corn for starch production (Maat et al. In Xylans and xylanases, eds. Visseret al., Elsevier Sci pub., Amsterdam. ISBN 0-444-894-772 (1992); McCleary, supra; Krishnarau et al., J. Food Sci. 59:1251-1254 (1994); U.S. Pat. No. 5,306,633); for use as animal food additives to aid in the digestibility of feedstuffs; and in the washing of super precision devices and semiconductors [Takayuki et al., U.S. Pat. No. 5,078,802). Several of these application could benefit from a thermostable xylanase, for example, food processing at elevated temperatures.
The active site of the Bacillus circulans xylanase is a wide cleft with two catalytic glutamates, E78 and E172, on either side and several aromatic tryptophan and tyrosine residues which act as binding sites for the substrate. The enzymatic mechanism consists of a nucleophilic attack of E78 on the 1,4-glycoside bond that is followed by a proton transfer from the acid/base catalyst E172 and a subsequent attack of a solvent water molecule where E172 now acts as a base. The enzymatic reaction results in retention of the configuration at the anomeric carbon [McCarter and Withers, Curr. Opin. Struc. Biol. 4:885-892 (1994)].
To this end, variants of xylanase (Xyn) sequences, applications and production procedures are known; see for example U.S. Pat. Nos.5,405,769; 5,736,384; 5,759,840; Arase et al. [FEB Lett. 316(2):123-7 (1993)]; Wakarchuket al.[Protein Sci. 3(3):467-75 (1994); Protein Eng. 7(11):1379-86 (1994)]; and references cited therein.
Recently, the crystal structures of recombinant Bacillus circulans xylanase [PDB entry 1XNB; Campbell et al., in Suominen and Geinikainen , eds. Proceedings of the second TRICEL symposium on Trichdema reesei cellulases and other hydrolases, Espoo, Finland, Helsinki: Foundation for Biotechnological and Industrial Fermentation Research, pp 63-77 (1993)1; expressly incorporated by reference) have been solved. In addition, structures for xylanases from Aspergillus niger Xyn A [Krengel and Dijkstra, J. Mol. Biol. 263(1):70-78 (1996); PDB entry 1ukr]; Paecilomyces varotii [J. Mo Biol. 243(4):806-808 (1994); PDB entry 1 PVX]; Trichoderma harzianum [Campbell et al., PDB entry 1XND]; Trichoderma reesei [Torronene and Rouvinen, Biochemistry 34:847 (1995); PDB entry 1XYN]; Trichoderma reesei [Torronene et al., EMBO J. 13(11):2493-2501 (1994); PDB entry 1ENX], all of which are expressly incorporated by reference. The three-dimensional structure of Bacillus circulans xylenase is composed of three beta-sheets and one alpha-helix. The first two beta-sheets (I and II) are roughly parallel, while the third one (sheet III) is at about a 90 degree angle to sheet II. Sheets I and II are each composed of five strands, while sheet III contains six strands. The alpha-helix lies across the back of sheet III and the last two strands of sheet III fold over one edge of the alpha-helix. The active site lies in the cleft between sheets II and III (PDB entry 1XNB; U.S. Pat. No. 5,405,769, herewith expressly incorporated as reference).
When carrying out protein engineering to modify protein properties, usually one had to select from the following options: (i) site-specific mutagenesis and (ii) random mutagenesis of the nucleic acid encoding the protein, or (iii) post-translational chemical modifications. No matter which method of protein engineering is used, a key aspect is determining which amino acids to modify, because few choices will improve the properties of the protein. The available crystal structure of xylanase allows a completely different approach by using computational protein design and the generation of more stable proteins or protein variants with an altered activity. Several groups have applied and experimentally tested systematic, quantitative methods to protein design with the goal of developing general design algorithms (Hellinga et al., J. Mol. Biol. 222: 763-785 (1991); Hurley et al., J. Mol. Biol. 224:1143-1154 (1992); Desjarlaisl et al., Protein Science 4:2006-2018 (1995); Harbury et al., Proc. Natl. Acad. Sci. U.S.A. 92:8408-8412 (1995); Klemba et al., Nat. Struc. Biol. 2:368-373 (1995); Nautiyal etal., Biochemistry 34:11645-11651 (1995); Betzo etal., Biochemistry 35:6955-6962 (1996); Dahiyat et al., Protein Science 5:895-903 (1996); Dahiyat et al., Science 278:82-87 (1997); Dahiyat et. al., J. Mol. Biol. 273:789-96; Dahiyat et al., Protein Sci. 6:1333-1337 (1997); Jones, Protein Science 3:567-574 (1994); Konoi, et al., Proteins: Structure, Function and Genetics 19:244-255 (1994)). These algorithms consider the spatial positioning and steric complementarity of side chains by explicitly modeling the atoms of sequences under consideration. In particular, WO98/47089, and U.S. Ser. No. 09/127,926 describe a system for protein design; both are expressly incorporated by reference.
A need still exists for xylanase enzymes exhibiting both significant thermostability, thermophilicity, alkalophilicity and xylanase activity. It is therefore an object of this invention, to provide novel xylanase activity (XA) proteins that are active at higher pH and temperature ranges as the naturally occurring xylanases. The novel XA proteins may find wider application in the pre-treatment of kraft pulp and other applications.
In accordance with the objects outlined above, the present invention provides non-naturally occurring xylanase activity (XA) proteins (e.g. the proteins are not found in nature) comprising amino acid sequences that are less than about 97% identical to Bacillus circulans xylanase. The XA proteins have at least one altered biological property when compared to Bacillus circulans xylanase; for example, the XA proteins will be more alkalophilic or more thermophilic or more thermostable or hydrolyze a substrate more efficiently than Bacillus circulans xylanase. Thus, the invention provides XA proteins with amino acid sequences that have at least about 3-5 amino acid substitutions as compared to the Bacillus circulans xylanase sequence shown in FIG. 1 (SEQ ID NO:1).
In a further aspect, the present invention provides non-naturally occurring XA conformers that have three dimensional backbone structures that substantially correspond to the three dimensional backbone structure of Bacillus circulans xylanase. The amino acid sequence of the XA conformer and the amino acid sequence of Bacillus circulans xylanase are less than about 97% identical. In one aspect, at least about 90% of the non-identical amino acids are in a core region of the conformer. In other aspects, the conformer have at least about 100% of the non-identical amino acids are in a core region of the conformer.
In an additional aspect, the changes are selected from the amino acid residues at positions selected from positions 7, 26, 28, 30, 39, 53, 58, 63, 64, 65, 67, 79, 80, 83, 84, 85, 88, 96, 98, 100, 102, 103, 105, 109, 110, 118, 128, 129, 130, 132, 136, 142, 144, 147, 148, 149, 150, 152, 156, 158, 160, 167, 168, 171, 176, 180, and 182. In a preferred aspect, the changes are selected from the amino acid residues at positions selected from positions 26, 28, 30, 53, 58, 64, 79, 105, 142, 171, 176, 180, and 182. In one aspect, the changes are selected from the amino acid residues at positions selected from positions 53, 83, 84, 85 105, 132, 136, 142, 144, and 149. In another aspect, the changes are selected from the amino acid residues at positions selected from positions 79, 96, 98, 100, 102, 103, 105, 109, 128, 130, 132, 144, 147, 148, 149, 150, 152, 156, 158, 160, and 167. In another aspect, the changes are selected from the amino acid residues at positions selected from positions 7, 39, 63, 65, 67, 88, 110, 118, 129, and 168. Preferred embodiments include at least about 3-5 variations.
In a further aspect, the invention provides recombinant nucleic acids encoding the non-naturally occurring XA proteins, expression vectors comprising the recombinant nucleic acids, and host cells comprising the recombinant nucleic acids and expression vectors.
In an additional aspect, the invention provides methods of producing the XA proteins of the invention comprising culturing host cells comprising the recombinant nucleic acids under conditions suitable for expression of the nucleic acids. The proteins may optionally be recovered.
In a further aspect, the invention provides a bleaching agent comprising as an active ingredient an XA protein.
In an additional aspect of the invention, the invention provides a method for bleaching pulp, said method comprising the step of contacting pulp to be bleached with the bleaching agent. The method may further comprise the step of chemical bleaching and/or an alkali extraction before, after or during said step of contacting pulp with said bleaching agent.